Mitochondrial Permeability Transition And Hepatitis B Virus Replication Study Of The Relationship

Hepatitis B virus (HBV) infection is a worldwide health problem, which still can't be healed well. HBV expresses a non-structural protein, which is very important for replication of HBV. HBx activates calcium-dependent Pyk2-Src signaling passway to increase HBV replication. It was reported that HBx protein colocalized to mitochondria with a human voltage-dependent anion channel, VDAC3, which was regarded as a component of mitochondrial permeability transition pore complex (PTPC). The mitochondrial permeability transition (MPT) is a sudden increase of the inner membrane permeability to solutes with molecular mass below approximately 1500 Da when the cells is in time of stress. Repression of HBV replication by cyclosporin A (CsA), a inhibitor of PTPC, was reported recently. Therefore, researchers hypothesize that HBx localizes on mitochondria and induce MPT to activate calcium-dependent Pyk2-Src signaling passway and HBV replication. However, direct evidence of HBx in physiological expression level inducing MPT is still absent. And CsA is not a specific inhibitor of PTPC, which binds to many targets in cells. In addition, suppression of HBV replication by blocking Ca²⁺ released from endoplasmic reticulum was reported recently. Therefore, the hypothesis above need more compact evidence. Here we use a new method to detect MPT and RNAi technology to discover the relation of between HBV replication and MPT.In the first chapter, the impact of HBV replication on the MPT was in HepG2 cell line by Calcein-AM/CoCl₂ staining, a new method to detect MPT directly. Induction of mitochondrial permeability transition by HBV replication in HepG2 cells is demonstrated. Then, HBx gene is constructed into pCMV-HA, an eukaryotic expression vector. Expression of HBx also induced MPT in HepG2 cells. Our result showed that mitochondrial permeability transition was induced by HBV replication in HepG2 cells, and associated with HBx protein.In the second chapter, in order to depress MPT specifically, esiRNAs of several components of PTPC were prepared. The segments of Ant1, Ant2, Vdac3 and CypD genes were cloned, and inserted respectively into expression vector with both forward and reversed direction with an interval sequence in between. Consequently the sense strand and anti-sense strand of the gene segments formed a long double-strand RNA(dsRNA) by complementary base pairing, followed transcription driven by T promoter. Those dsRNAs of four different genes were cleaved respectively by E.coli derived RNase III in the presence of Mn²⁺, and esiRNAs of 21 bp in length were purified. HepG2 cells were transfected with these four esiRNAs or control esiRNA. Semi-quantitative RT-PCR were used to tested the efficiency and specificity of different esiRNA on mRNA level. The result showed that the esiRNA of four genes only induced degradation of mRNA of the target genes, while they have no effect on the mRNA level of other homologues genes. In order to test the expression of other homologues genes can blocked by esANT1 or not. EsiANT1 target sequence of Ant1 and coordinately sequences from Ant2 and Ant3 genes was separately inserted into the 3' UTR of EGFP reporter gene. CHO cells were co-transfected with these reporter plasmids and either esiANT1 or chemical synthesized siANT1. Expression level of EGFP was quantified by flow cytometer to find whether esiRNA may interfere the expression of the highly homological Ant2 and Ant3 genes. The result demonstrated that esiANT1 had the same effect as chemical synthesized siANT1 on blocking the expression of EGFP which containing part of Ant] sequence at its 3' UTR, while it has no discemable interference effect on the expression of EGFP that containing part of Ant2 or Ant3 sequence at its 3'UTR. So it is showed that esiRNA had high efficiency, as well as enough specificity.In the third chapter, HepG2 cells were co-transfected with pUC-HBV2 plasmid containing two copies of HBV genome, and esiANT1, esiANT2, esiVDAC3 or esiCypD respectively. By CalceinAM/CoCl₂ staining, it was showed that only esiCypD down-regulated the PTPC openning level induced by HBV replication. Then esiCypD was introduced into 2.2.15 cell line, which can stably produce HBV particles, and also down-regulated HBV-replication-dependent MPT. was HepG2 cells were co-transfected with pUC-HBV2 and these four esiRNA respectively. Expression of surface antigen and e antigen of HBV , as well as replication of HBV DNA was quantified subsequencly. The result showed that the four kinds of esiRNA didn't affect the expression of HBV S antigen and e antigen, but esiANT1,esiANT2 and esiCypD had suppression effect on HBV's DNA replication.In conclusion, this dissertation demonstrated directly for the first time that HBV replication could induce MPT, associated with HBx protein. Decrease of PTPC opening by RNAi could reduce HBV-induced MPT, and repression DNA replication of HBV.